Paraffin Technique, Cells, Epithelium Flashcards
INSTRUMENTS USED IN THE STUDY
OF HISTOLOGY
- TISSUE CASSETTE/MOULDER
- PARAFFIN DISPENSER
- TISSUE BLOCK CUTTER
- ROTARY MICROTOME
- MICROTOME BLADES
- TISSUE FLOATER
- TISSUE SLIDE DRYER
- MICROSCOPE
- AUTOMATIC PROCESSOR
- H & E STAIN
➢ Acquisition of tissue samples
• From the slaughterhouse or thru biopsy
• The most critical step (time)
• Size of tissue to be collected –
match box
➢Fixation of tissue samples
Fixatives denature the protein contents and inactivates the
enzymes of the tissue thereby rendering a almost in vivo condition
Formaldehyde, picric acid, osmic acid, mercuric chloride, acetone
are common simple fixative
However, no single fixative is commonly used
10% NBF is the most commonly used
Length of fixation depends on the size of the tissue
➢ Washing of fixed tissues
• Removal of excess fixative through running water
➢Dehydration of washed tissue
• Immersion of washed tissue to increasing concentration of alcohol (70-100% ethyl alcohol) to remove excess water.
➢Clearing of dehydrated tissue
The removal of alcohol that has been introduced during dehydration
Alcohol is not miscible with the paraffin embedding medium and should be removed
Xylene is the most commonly used clearing agent
➢Impregnation/infiltration of cleared tissue
•Immersion of cleared tissue to paraffin solution that may permit cutting of thin sections w/out damage to tissue and
its cellular components
➢Embedding/casting/blocking
•Impregnated tissue is placed in molder or boat filled with melted paraffin and allowed to solidify to form block
➢Trimming of tissue blocks
•Paraffin blocks are then cut of excess paraffin to expose the tissue surface in preparation for actual
cutting
➢Sectioning of tissue blocks
•The cutting of tissue block into thin sections ( 5-7 microns) with the aid of a microtome, sections tend to form tissue ribbons
➢Mounting of tissue sections into glass slides
•Tissue” ribbons” are floated in warm water bath to stretch or flatten them
•Selected sections are then mounted onto a glass slides and allowed to dry ( air or oven)
The removal of paraffin from the tissue sections by immersion to xylene.
Deparaffinization
Introduction of water to deparaffinized sections through decreasing concentrations of alcohol (100-70%) to water in preparation for staining.
Rehydration
Routinely used is the H & E stain
- Basic dye Hematoxylin react with the acidic components of the cell, the nucleus which stains blue
- Acidic dye Eosin react to the basic cellular components of the cell, the cytoplasm that colors pink to red
Staining with acid/base dyes
Staining Process
- Deparaffinization
- Rehydration
- Staining with acid/base dyes
- Washing
- Dehydration
- Clearing
- Mounting
The smallest and basic unit structure of a protoplasm that can exist independently.
Cell
The collective term for the living substance of the cell of a plant or an animal.
Protoplasm
The sum of all chemical reactions occurring
within the cell.
Metabolism
A reaction resulting from the synthesis of new molecular components essential for growth, maintenance and repair
Anabolism
A reaction resulting to the degradation of cellular components with the release of energy
Catabolism
The ability of the cell to respond to stimuli in their
environment
Irritability
The ability of the cell to shorten along their long axes (special property of muscle)
Contractility
The ability of the cell to elaborate useful new substances.
Example - secretion of the hepatocytes of bile, pancreatic juice by the pancreas
Secretion
The ability of the cell to get rid of the waste product of metabolism.
Ex. Urine by the kidney, feces by the GIT
Excretion
The ability of the cell to transmit impulses along their cell membrane (special property of nerve cells)
Conductivity
A process by which materials gain access to the cell either by diffusion or pinocytosis
Endocytosis
“Pinching off of the cell membrane”.
Pinocytosis
Engulfment and uptake of particulate matter
Phagocytosis
The exit of materials from the cell
Exocytosis
Cell size varies per cell
❑ Sperm 3-5 µm
❑ RBC 5 – 7 µm
❑ Hepatocyte 30 – 50 µm
❑ Egg cell – 100 µm
CELL SIDES/SURFACES
❑ Apical
❑ Lateral
❑ Basal
❖ Polarized
Cell is divided into:
❑ Cytoplasm
❑ Nucleus
o Number varies from 1,2 or several to none at all as in the case of
mammalian RBC
o Shape also varies from round,
crescent shape or lobulated as in the
case of some WBC
o Position varies also from central,
paracentral to eccentrical location
o An interphase nucleus has four distinct
components:
• Nuclear membrane or envelope
• Karyolymph or nuclear sap
• Chromatin
• Nucleolus
Nucleus
A darkly basophilic membrane
▪ ultrastructurally, an outer and inner membrane exists separated by a
perinuclear space or cisterna
▪ perinuclear space and outer
membrane are contious with the RER
Nuclear membrane
The matrix of the nucleus and the soluble
phase of the nuclear material
Nuclear sap or karyolymph
A term used to describe any area in the nucleus suspected to contain DNA and its bound proteins
Chromatin
The condensed form of chromatin which stains deeply with basic dyes and is metabolically inert
Heterochromatin
A distinguishable heterochromatin believed to contain
the X chromosome
Barr body
non- membrane bound component of the nucleus which may occur freely or attached to the inner nuclear
membrane
✓composed of granular and
filamentous materials both of
which contain RNA
✓Is the center for the synthesis of
RNA (ribosomes)
✓May occur singly or more in the
cell and this determines the
malignancy of a cell
Nucleolus
Gen’ly is acidophilic composed
of three major constituents:
• grounsubstance/hyaloplasm/cytoplasm matrix,
• organelles and
• inclusion bodies
Cytoplasm
An admixture of H2O, CHON,CHO, organic and inorganic salts
Ground substance
The only non-membranous cell organelle
•synthesize proteins for intracellular use
• are RNA containing bodies in the nucleus and cytoplasm of the cell
• in the nucleus, they are found in the nuclear matrix of the nucleolus
• in the cytoplasm, they occur as free ribosomes individually or in groups called polyribosomes or polysomes or attached to the RER
Ribosomes
The membranous organelles :
▪ Cell membrane/plasma memb/ plasmalemma and cell coat
▪ Endoplasmic reticulum (smooth & rough)
▪ Golgi apparatus
▪ Mitochondria
▪ Lysosomes
25-75% of the weight of cell membrane
Distinguished according to function
❑ Structural proteins
❑ Ectoenzymes
❑ Transportation proteins
❑ Receptors
Membrane Protein
2-10% of the weight of cell membrane; covalently bound to
membrane proteins (glycoproteins) or membrane lipids (glycolipids)
Membrane Carbohydrates
All the carbohydrate chains at the surface of a cell
❑ Location of blood group properties
❑ Binding site for pathogens and toxin
❑ Cell-cell recognition
❑ Cell adhesion
Glycocalyx
A system of hollow structures either
tubules or flattened vesicles
(cisterna) extending throughout
the cytoplasm.
Endoplasmic Reticulum
Continuous with the nuclear
membrane studded with ribosomes (appears basophilic due to ribonucleoproteins )
Functions:
1. Protein synthesis destined for secretion
( e.g. digestive enzyme)
2. Glycogen biosynthesis
3. prod’n of degrative enzymes e.g.
glucose-6- PO4
Rough Endoplasmic Reticulum
–not studded with ribosomes, thus has no distinct staining characteristics
- less extensive than RER except in
certain cells like hepatocytes
Functions:
1. Participates in glycogen metabolism,
synthesis
2. Participates in ion concentration,
distribution and detoxification of certain
substances
3. Believed to contribute to the formation of
golgi apparatus
Smooth Endoplasmic Reticulum
- has a lamellated profile (4 or more) usually dilated sacks or cisternae; has 2 faces:
- convex face or immature/ formative/trans face- closely associated with transfer vesicle from RER
- concave or mature/open/cis face- intimately associated with secretory vesicles in various stages of condensation and maturation
Functions:
1. Participates in the dynamic turnover of cell membrane
2. Participates in the concentration and packaging of secretory products
3.synthesize polysaccharides
4. Repository of vitamin C
5. Participates in the formation of lysosomes
Golgi apparatus
•appears as thread or granules which could be stained specifically with
special stain called janus green B
• number varies in the cell depending on the energy requirements of the cell
•ultrastucturally, mitochondria appears as double membrane structure composed of outer and inner walls
• inner wall forms plate like or tubular folds called mitochondrial cristae
Functions:
1. Site of kreb’s citric acid cycle
2. Contains enzymes for oxidative
phosphorylation and fatty acid oxidation
3. Contains DNA & RNA thus possessing
some genetic and protein synthetic potentials
Mitochondria
▪ this membrane bound particles
contain hydrolytic enzymescollectively called acid hydrolases
▪ specific granules of the basophils,
eosinophils and neutrophils are
representative of lysosomes believed
to come from golgi apparatus as
primary or inactive lysosomes
Functions:
1. intracellular digestion of materials
taken in by the cells
2. autolysis
3. post mortem degeneration
Lysosome
- are two small bodies contained within an area of the cytoplasm known as centrosphere
- ultra-structurally, are seen as two cylindrical unit oriented to each other
- each cylinder is composed of nine groups of three tubules
Centrioles
- composed of proteins, tubulin which are similar to the action of muscle cell: are found in cilia, flagella and spermatids
- ultra-structure of cilia is composed of 9 doublets (axonemes) connected by nexin and 2 microtubule singlet
• A microtubule (13 protofilaments; complete)
•
B microtubule (11 protofilaments; incomplete)
Microtubules
❑ Either absorbed or generated
❑ Pigments, reserved and stored
materials
Inclusion bodies
finger-shaped; strengthened by 20-30 actin filaments; immobile or passively mobile; brush border
(intestinal cells)
Microvilli
long; finger-shaped; apical border of
columnar cells (epididymal epithelium and hair cells in inner
ear)
Stereocilia/villi
ridge-like folds (lateral or apical cell membrane)
Microplicae
motile; lash-shaped; can occur singly (spermatozoa) or in complexes (epithelium of fallopian tube and trachea)
Cilia
mechanically attaching cells to other cells or to extracellular matrix
Anchoring Junction
button-like cell-cell contacts occur in epithelial cells. (Intermediate filaments)
Desmosome (macula adhaerens)
connection of cells to the extracellular matrix (actin filaments)
Hemidesmosome
mechanically attaching cells
to other cells or to ECM
Anchoring Junction
belt desomosome; with bundles of parallel actin filaments
Adhesion junction- (zonula adhaerens)
Integral membrane proteins which form an extracellular connection with the same (homophile) or other
(heterophile) integral membrane proteins
Adhesion Proteins
It can adhere only in the presence of Calcium
Cadherin
The glucose-binding protein
Selectin
It allows cell to adhere to ECM
Integrin
Transcellular
protein channels composed of 6 connexins
Function - Metabolic coordination,
electrical coupling
Connexon (hemichannel)
Strands of globular transmembrane protein sealing the intercellular gap
Zonula Occludens (Tight Junction)
Internal supporting and movement apparatus
Function:
❑ Modification and maintenance of cell shape
❑ Specialized cell appendages
❑ Directed intercellular transportation
❑ Positioning of the cell nucleus and other organelles
❑ Cell motility
❑ Cell division
CYTOSKELETON
❑ 5-8nm thick
❑ 5-20% of total protein; present in all cells
❑ Actin is the basic unit
❑ Globular actin — Filamentary actin
Actin Filaments
❑ 8-10nm thick
❑ Protein subunit with tail and head
section
❑ Monomer- dimer- tetramer—
intermediate filament
❑ Globular actin — Filamentary actin
Intermediate Filaments
❑ 25nm thick
❑ α and β-tubulin dimers
❑ 13 protofilaments— microtubule
Tubulin is highly conserved protein
Microtubules
Specialization of cells
• Muscle cell – contraction
• Nerve cells – impulse transmission
• Gland cells – synthesis and discharge of enzymes
• Macrophage – intracellular digestion
Differentiate into any cell type
Totipotent
Differentiate into cells from any of the three germ layers
Pluripotent
❑ Extracellular matrix
- Collagen fibers, interstitial fluid
❑Basement membrane
INTERCELLULAR SUBSTANCE
•A group of more or less similar cells and their extracellular products
performing a variety of related functions
TISSUE
Four basic type of tissue
o Epithelial tissue
o Connective and supportive tissue
o Muscle tissue
o Nervous tissue
Living components of the tissue
Cell
Non- living materials produced by the cells
Intercellular substance
Liquid component of the tissue located between cells.
▪ Cerebro-spinal fluid
▪ Synovial fluid
▪ Blood
Fluids
Fibrous in nature
• Collagen
• Elastic
• Reticular fibers
Formed substance
A semi solid to firm gel in consistency
Amorphous substance
Free border
▪ Apical or luminal
Surface in contact with adjacent cells
▪Lateral
Border in contact with the underlying basal lamina
▪Basal
A tissue composed of closely aggregated cells in opposition over a
large part of their surfaces and having a very little intercellular space.
EPITHELIAL TISSUE
No blood supply of its own
Avascular
Three Major types of Epithelial Tissue
1.Covering (surface) and lining epithelium-
2.Special epithelium
3.Glandular epithelium
Basis in naming epithelium
▪ Shape of surface cell
▪ Number of cell layer
Contain only one layer of cell
Simple epithelium
Has two or more layers of cell
Stratified epithelium
(false) consists only of a single layer of that rest on the basal lamina but not all reaching the apical surface.
Cells reaching the apical surface are tall columnar cells with nuclei located on a higher level while those not reaching the surface are shorter cells with nuclei located on a lower level,
thus, appearing multi layered
Pseudostratified epithelium
Cells are polygon on surface
view and thin, elongated on longitudinal view
Squamous epithelium
Short, as tall as they are wide
Cuboidal epithelium
Rectangular, taller than they are wide
Columnar epithelium
Cytoplasm is sparse, only nucleus is
visible
Simple squamous epithelium
Cytoplasm is sparse, only nucleus is
visible
Simple squamous epithelium
It present four equal sides with
round nuclei located at
the center of the cells
Simple cuboidal epithelium
- Composed of taller than are wide
cells with oval nuclei located parabasally - Other cell types are present
(goblet cells, hormone-producing cells) - Evident microvilli
Simple columnar epithelium
▪found in large ducts of exocrine
glands
Simple clumnar epithelium with
typical columnar cells
▪ Cells are also similar but differes from the typical columnar cells by
their vacoulated cytoplasm due to dissolution of mucin granules during paraffin technique
▪ Found the stomach, abomasum
and cervix
SCE with mucous secreting columnar cells
• the secretory cell is the goblet and the absorptive is a typical columnar cell that have microvilli (tiny finger-like projections) on their
apical/ luminal borders
•Found in the small and large intestine
Simple columnar with both secretory and absorptive cells
Columnar cells present cilia (hair like projections in their apical borders) instead of microvilli.
▪ Found only in trachea
▪ Also known as the pseudostratified columnar ciliated epithelium
Simple columnar epithelium with both secretory and ciliated cells
– (false) only of a single layer of cell that rest on the basal lamina — but not all reach the apical surface.
- cells reaching the apical surface are tall columnar cells with nuclei located on a higher level
- while those not reaching the surface are shorter cells with nuclei located on a lower level, thus, appearing multi layered
Pseudostratified columnar epithelium
- pillow-shaped surface epithelial cell (=covering cells)
- intermediate cells
- 1-2 basale layers (cuboidal to columnar cells)
- description as pseudostratified or stratified
Transitional epithelium
Rounded surface cells
Unstretched
Flattened and elongated cells
Stretched
Classification; Number of cell layer
❑Stratified Epithelium
• Stratified Squamous keratinized
• Stratified Squamous non-
keratinized
• Stratified Cuboidal
• Stratified Columnar
❑ two or more layers of cells
❑ Classification depends on the shape of
the outermost cells
STRATIFIED EPITHELIAL
TISSUE
■ composed of several layers of cells: deepest cells are
columnar or cuboidal cells resting on the basement membrane;
■ middle layer are polyhedral or polygonal cells
■ and the superficial layer are squamous cells;
■ lines the inner surfaces of the body exposed to considerable wear and tear such as the esophagus, vagina etc ;
■ covers the outer surfaces of the body such as the epidermis of the skin;
STRATIFIED SQUAMOUS EPITHELIUM NON KERATINIZED
- covers also the outer surfaces of the body such as the epidermis of the skin;
- superficial cells undergo metamorphosis into tough non-living layer of keratin
- Keratin prevents fluid from evaporating and keeps the body from absorbing water when one is taking a bath,
-impervious to bacteria- hence a first line of defense against infection
Stratified squamous epithelium keratinized
- Composed of two layers only: basal layer of cuboidal cells and superficial columnar cells
- found only in the fornix of the conjunctiva, cavernous urethra; large excretory ducts of exocrine glands
STRATIFIED COLUMNAR EPITHELIUM
Function of Surface Epithelium
❑ Protection– skin
❑ Absorption– active or passive absorption (intestine)
❑ Transport– epididymis, trachea
❑ Excretion – ureter, urethra
Increase absorption surface (intestinal epithelium)
Microvilli
long microvilli; non-motile (epididymis; haircells)
Stereocilia/villi
motile (tracheal and bronchial epithelium; uterine epithelium)
Kinocilia (brush border)
A unit of cells, which are specialised for the synthesis and secretion of secretory products (secretions).
Glandular Epithelium
Glands which secret out of the body and therefore are provided with ducts
Exocrine
Glands w/out ducts or glands of internal secretion, secretions poured into closely associated capillaries
Endocrine
Location of glandular epithelium
- Intraepithelial
■ Unicellular– goblet cells (intestinal epithelium)
■ Multicellular– epithelium of nasal cavity - Extraepithelial– located in the underlying connective tissue, always multicellular, secretion is produced in secretory units and reach by a duct system
Shaped of secretory units
• Simple - unbranched duct, which may be straight or coiled
• Compound - shows a repeated branching pattern of the ducts
Methods of Secretion
- Endocrine glands – secrete products directly into the
surrounding connective tissue
■ Paracrine – target cell is nearby
■ Autocrine – target is the same cell - Exocrine glands – secretes product through a duct
Mode of Secretion
a) Merocrine (eccrine) – released by exocytosis (sweat glands, salivary gland)
b) Apocrine – released by apocytosis (sweat glands)
c) Holocrine – whole cell (sebaceous gland)
Nature and Type of Secretory Product
❑Serous glands – pyramid-shape, narrow lumen, produce watery thin secretion; rich in protein and enzymes
❑Mucous glands – wife lumen, produce thick, viscous product; rich in mucin
❑Mixed glands- serous “end pieces” are lying cap-shaped on the mucus ones= serous Gianuzzi-demilunes, secrete combination of serous and mucous
